Compounds having affinity for the granulocyte-colony stimulating factor receptor (G-CSFR) and associated uses

ABSTRACT

Novel compounds are provided that bind to G-CSFR. The novel compounds have a peptide chain approximately 6 to 40 amino acids in length that binds to G-CSFR. The compounds are useful as probes for affinity screening. In addition, the compounds have demonstrated agonist or antagonist activity for the G-CSFR, and are therefore useful in treatment of diseases including patients who suffer from a low white blood cell titer. Pharmaceutical compositions and methods of use are provided as well.

TECHNICAL FIELD

The present invention relates generally to novel compounds that haveaffinity for the granulocyte-colony stimulating factor receptor(G-CSFR). More particularly, the invention relates to such compoundswhich act as G-CSF mimetics by activating or inactivating the G-CSFR, orby affecting ligand binding to G-CSFR. The invention additionallyrelates to methods of using the novel compounds and pharmaceuticalcompositions containing a compound of the invention as the active agent.The invention has application in the fields of biochemistry andmedicinal chemistry and particularly provides G-CSF mimetics for use inthe treatment of human disease.

BACKGROUND

Granulocyte-colony stimulating factor (G-CSF) is a hematopoietic growthfactor that specifically stimulates proliferation and differentiation ofcells of the neutrophilic lineage.

G-CSF is a cytokine that binds to and activates the granulocyte-colonystimulating factor receptor (G-CSFR). G-CSFR is expressed on the surfaceof mature neutrophils and cells committed to the neutrophilic lineage,with receptor density varying from 190 to more than 1400 sites per cell.The receptor is a member of the cytokine receptor superfamily; itcontains a cytokine receptor-homologous domain responsible for G-CSFbinding, an immunoglobulin-like domain, three fibronectin type IIIdomains, a transmembrane region, and an intracellular domain. Theobserved affinity of G-CSF for its receptor is about 100 pM.

The complete G-CSF protein has become an important therapeutic agent inclinical indications involving depressed neutrophil counts. Suchindications include chemotherapy-induced neutropenia, AIDS and communityacquired pneumonia. Furthermore, G-CSF antagonists may be useful in thetreatment of some diseases caused by an inappropriate or undesirableactivation of G-CSFR.

There remains a need, however, for compounds that bind specifically toG-CSFR, both for studies of the important biological activities mediatedby the receptor and for treatment of diseases, disorders and conditionsthat would benefit from activating or inactivating G-CSFR. The presentinvention provides such compounds, and also provides pharmaceuticalcompositions and methods for using the compounds as therapeutic agents.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides compounds comprising a peptidechain that binds to G-CSFR. In one aspect, the peptide chain isapproximately 10 to 40 amino acids in length and contains a sequence ofamino acids of formula (I) (I) CX₁X₂X₃X₄X₅X₆X₇X₈C (SEQ ID NO: 1)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X₁ is A, N, S, F, D, G, L, T, E, V, P, Q, H, Mor K; X₂ is M, G, R, H, D, I, V, A, S, E, N, F, Y, P, C, W or T; X₃ isE, V, W, F, M, A, N, S, L, T, Y, G or P; X₄ is V, I, G, Q, W, M, T, Y,L, P, D, C, E or A; X₅ is M, E, W, L, P, N, I, T, V, F, Y, Q, S, R, W,G, H or D; X₆ is H, A, W, Y, V, F, Q, M, N, E, S, D, P or G; X₇ is M, F,Y, V, N, L, H, D, S, W, G, Q, C or T; and X₈ is C, Y, R, I, K, W, L, E,M, H, A, T, F, D, P, G or Q.

In another aspect, the peptide chain is approximately 9 to 40 aminoacids in length and contains a sequence of amino acids of formula (II)(II) X^(I) ₁X^(I) ₂X^(I) ₃SGWVWX^(I) ₄ (SEQ ID NO: 2)wherein each amino acid is indicated by the standard one-letterabbreviation, and wherein X^(I) ₁ is S, Q, R, L or Y; X^(I) ₂ is N, S,T, A or D; X^(I) ₃ is E, D or N; and X^(I) ₄ is L V, T, P or H.

In another aspect, the peptide chain is 6 to 40 amino acids in lengthand contains a sequence of amino acids of formula (III) (III) ERX^(II)₁X^(II) ₂X^(II) ₃C (SEQ ID NO: 3)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(II) ₁ is D, L, S, G, E, A, K or Y; X^(II) ₂is W, Y, F, L or V; and X^(II) ₃ is F, G, M or L.

In still another aspect, the peptide chain is approximately 9 to 40amino acids in length and contains a sequence of amino acids of formula(IV) (IV) X^(III) ₁MVYX^(III) ₂X^(III) ₃PX^(III) ₄W (SEQ ID NO: 4)wherein each amino acid in indicated by standard one-letterabbreviation, and wherein X^(III) ₁ is D or E; X^(III) ₂ is A or T;X^(III) ₃ is Y or V; and X^(III) ₄ is P or Y.

In an additional aspect, the invention provides compounds comprising apeptide chain approximately 12 to 40 amino acids in length and containsa sequence of amino acids of formula (V) (V) CX^(IV) ₁X^(IV) ₂X^(IV)₃X^(IV) ₄X^(IV) ₅X^(IV) ₆X^(IV) ₇X^(IV) ₈X^(IV) ₉X^(IV) ₁₀C (SEQ ID NO:5)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(IV) ₁ is E, G, P, N, R, T, W, S, L, H, A, Qor Y; X^(IV) ₂ is S, T, E, A, D, G, W, P, L, N, V, Y, R or M; X^(IV) ₃is R, Y, V, Q, E, T, L, P, S, K, M, A or W; X^(IV) ₄ is L, M, G, F, W,R, S, V, P, A, D, C or T; X^(IV) ₅ is V, T, A, R, S, L, W, C, I, E, P,H, F, D or Q; X^(IV) ₆ is E, Y, G, T, Q, M, S, N, A or P; X^(IV) ₇ is C,V, D, G, L, W, E, V, I, S, M or A; X^(IV) ₈ is S, Y, A, W, P, V, L, Q,G, K, F, I, E or D; X^(IV) ₉ is R, W, M, D, H, V, G, A, Q, L, S, E or Y;X^(IV) ₁₀ is M, L, I, S, V, P, W, F, T, Y, R, or Q.

In another aspect the peptide chain is approximately 9 to 40 amino acidsin length and contains a sequence of amino acids of formula (VI) (VI)X^(V) ₁X^(V) ₂X^(V) ₃X^(V) ₄X^(V) ₅X^(V) ₆CX^(V) ₇X^(V) ₈ (SEQ ID NO: 6)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(V) ₁ is E, C, Q, V, or Y; X^(V) ₂ is E, A,L, M, S, W, or Q; X^(V) ₃ is K, R or T; X^(V) ₄ is L, A, or V; X^(V) ₅is R, A, M, H, E, V, L, G, D, Q, or S; X^(V) ₆ is E or V; X^(V) ₇ is Aor G; X^(V) ₈ is R, H, G or L.

In a further aspect, the peptide chain is approximately 10 to 40 aminoacids in length that binds to G-CSFR and contains a sequence of aminoacids of formula (VII) (VII) X^(VI) ₁X^(VI) ₂X^(VI) ₃X^(VI) ₄X^(VI)₅EX^(VI) ₆X^(VI) ₇X^(VI) ₈X^(VI) ₉ (SEQ ID NO: 7)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(VI) ₁ is A, E or G; X^(VI) ₂ is E, H or D;X^(VI) ₃ is R or G; X^(VI) ₄ is K, Y, M, N, Q, R, D, I, S or E; X^(VI) ₅is A, S or P; X^(VI) ₆ is E, D, T, Q, K or A: X^(VI) ₇ is R, W, K, L, S,A or Q; X^(VI) ₈ is R or E; and X^(VI) ₉ is W, G, or R.

In a final aspect, the invention also provides peptides that, while notnecessarily corresponding to one of the above-defined formulas, bind toG-CSFR.

In some contexts, the compounds of the invention are preferably in theform of a dimer. It is also preferred, in some contexts, that thecompounds of the invention include a peptide wherein the N-terminus ofthe peptide is coupled to a polyethylene glycol molecule. In somecontexts, it is preferred that the compounds of the invention include apeptide wherein the N-terminus of the peptide is acetylated. Inaddition, it is preferred, in some contexts, that the compounds of theinvention include a peptide wherein the C-terminus of the peptide isamidated.

The invention also provides a pharmaceutical composition that comprisesa therapeutically effective amount of a compound of the invention incombination with a pharmaceutically acceptable carrier, as well as amethod for treating a patient who would benefit from a G-CSFR modulator,the method comprising administering to the patient a therapeuticallyeffective amount of a compound of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 and 1-11 providethe sequences of representative peptide chains contained within thecompounds of the invention.

FIGS. 2, 3, 4, 5, 6, 7, 8, 9A, 9B 10A, 10B and 11 are graphs showing theresults of various assays described in Examples.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions and Overview

It is to be understood that unless otherwise indicated, this inventionis not limited to specific peptide sequences, molecular structures,pharmaceutical compositions, or the like, as such may vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a novel compound” in a pharmaceutical composition meansthat more than one of the novel compounds can be present in thecomposition, reference to “a pharmaceutically acceptable carrier”includes combinations of such carriers, and the like.

In this specification and in the claims that follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G. In addition, “1-Nal” is used to refer to1-naphthylalanine, the “2-Nal” is used to refer to 2-naphthylalanine.

Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic compounds of the present invention. Examplesof unconventional amino acids include: β-alanine, 1-naphthylalanine,2-naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,nor-leucine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline).

“Peptide” or “polypeptide” refers to a polymer in which the monomers arealpha amino acids joined together through amide bonds. Peptides are twoor often more amino acid monomers long. One or more of the peptidechains disclosed herein may appear in the compounds of the present. Itis also contemplated that the peptide chains disclosed herein representonly a portion of the overall peptide included in the compound.

The term “dimer” as in a peptide “dimer” refers to a compound in whichtwo peptide chains are linked; generally, although not necessarily, thetwo peptide chains will be identical and are linked through a linkingmoiety covalently bound to the carboxyl terminus of each chain.

The term “agonist” is used herein to refer to a ligand that binds to areceptor and activates the receptor.

The term “antagonist” is used herein to refer to a ligand that binds toa receptor without activating the receptor. Antagonists are eithercompetitive antagonists or noncompetitive antagonists. A “competitiveantagonist” blocks the receptor site that is specific for the agonist. A“noncompetitive antagonist” inactivates or otherwise affects thefunctioning of the receptor by interacting with a site other than theagonist binding site.

The term “modulator” as in a “G-CSFR-modulator” refers to a compoundthat is either an agonist or antagonist of the G-CSFR.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult can be alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system.Preferably, this dose or amount will be sufficient to either at leastpartially activate or at least partially inactivate G-CSFR and, thus,alleviate the symptoms associated with an undesired neutrophil count invivo.

An “optimal neutrophil count” refers to a quantity of neutrophils in apatient that is determined by a clinician to be optimal for that patientin light of the patient's disease state, condition, etc.

An “undesired neutrophil count” refers to a quantity of neutrophils in apatient that is determined by a clinician to be not optimal for thatpatient in light of the patient's disease state, condition, etc. Thus,an undesired neutrophil count may be depressed, elevated or even equalto the expected neutrophil count so long as the clinician determinesthat the actual count is not optimal for the patient. The compounds ofthe present invention are intended to, inter alia, provide the clinicianwith compounds that, when administered to a patient, bring thatpatient's neutrophil count closer to an optimal neutrophil count.

The term “treat” as in “treat a disease” is intended to include anymeans of treating a disease in a mammal, including (1) preventing thedisease, i.e., avoiding any clinical symptoms of the disease, (2)inhibiting the disease, that is, arresting the development orprogression of clinical symptoms, and/or (3) relieving the disease,i.e., causing regression of clinical symptoms.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

By “pharmaceutically acceptable carrier” is meant a material which isnot biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected active agentwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

II. The Compounds

A. Compounds of Formula (I):

In a first embodiment, the invention provides compounds comprising apeptide chain that binds to G-CSFR, wherein the compounds comprise apeptide chain approximately 10 to 40 amino acids in length that binds toG-CSFR and contains a sequence of amino acids of formula (I) (I)CX₁X₂X₃X₄X₅X₆X₇X₈C (SEQ ID NO: 1)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X₁ is A, N, S, F, D, G, L, T, E, V, P, Q, H, Mor K; X₂ is M, G, R, H, D, I, V, A, S, E, N, F, Y, P, C, W or T; X₃ isE, V, W, F, M, A, N, S, L, T, Y, G or P; X₄ is V, I, G, Q, W, M, T, Y,L, P, D, C, E or A; X₅ is M, E, W, L, P, N, I, T, V, F, Y, Q, S, R, W,G, H or D; X₆ is H, A, W, Y, V, F, Q, M, N, E, S, D, P or G; X₇ is M, F,Y, V, N, L, H, D, S, W, G, Q, C or T; and X₈ is C, Y, R, I, K, W, L, E,M, H, A, T, F, D, P, G or Q.

Preferably X₁ is D or P; X₂ is D or P; X₃ is E or W; X₄ is V, I or Y; X₅is M or L; X₆ is W, Y or F; X₇ is M, Y or D; and X₈ is C or M.

Examples of particularly preferred sequences satisfying formula (I)include, but are not limited to, the following: CAGEVMHMCC; (SEQ ID NO:8) CNREIEAMCC; (SEQ ID NO: 9) CADEVMHFCC; (SEQ ID NO: 10) CNREIMWMCC;(SEQ ID NO: 11) CSHEVWWYCC; (SEQ ID NO: 12) CSREVLYYCC; (SEQ ID NO: 13)CFIEGPWVCC; (SEQ ID NO: 14) CFVEGNWYCC; (SEQ ID NO: 15) CAAEVMVNCC; (SEQID NO: 16) CSDEVIFYCC; (SEQ ID NO: 17) CDREIMWFCC; (SEQ ID NO: 18)CAHEVMWMCC; (SEQ ID NO: 19) CGSEVTFMCC; (SEQ ID NO: 20) CLEEIMWLCC; (SEQID NO: 21) CAREVLAMCC; (SEQ ID NO: 22) CSVEVMQMCC; (SEQ ID NO: 23)CTNVQLMHYC; (SEQ ID NO: 24) CDVWQLFDRC; (SEQ ID NO: 25) CSFVQLNSIC; (SEQID NO: 26) CDYWQWFDKC; (SEQ ID NO: 27) CESFWVELWC; (SEQ ID NO: 28)CVPWMFYDLC; (SEQ ID NO: 29) CDPWMFYDLC; (SEQ ID NO: 30) CDPWVLFDEC; (SEQID NO: 31) CDHWTYFDMC; (SEQ ID NO: 32) CVVWTLYDKC; (SEQ ID NO: 33)CPDWYQSYMC; (SEQ ID NO: 34) CPDWYSYYMC; (SEQ ID NO: 35) CPEWYTDVMC; (SEQID NO: 36) CPDWYLDYMC; (SEQ ID NO: 37) CPEWYLDYMC; (SEQ ID NO: 38)CPDWYLPYMC; (SEQ ID NO: 39) CPEWYLPYMC; (SEQ ID NO: 40) CQDWWVELWC; (SEQID NO: 41) CPDWYLPWMC; (SEQ ID NO: 42) CACMLRVVHC; (SEQ ID NO: 43)CQRAGYMLAC; (SEQ ID NO: 44) CHANPVWGEC; (SEQ ID NO: 45) CFWSDWGQTC; (SEQID NO: 46) CPHWTSYYMC; (SEQ ID NO: 47) CETLCGACFC; (SEQ ID NO: 48)CATTINDTLC; (SEQ ID NO: 49) CLNYPHPVFC; (SEQ ID NO: 50) CMDGEMAVDC; (SEQID NO: 51) CNMGWMSWPC (SEQ ID NO: 52) CETYADWLGC; (SEQ ID NO: 53)CDPWMFFDMC; (SEQ ID NO: 54) CDPWIWYDLC; (SEQ ID NO: 55) CDPWIMYDRC; (SEQID NO: 56) CDPWVFFDIC; (SEQ ID NO: 57) CDPWTYYDLC; (SEQ ID NO: 58)CDPWIFYDRC; (SEQ ID NO: 59) CDPWLFYDLC; (SEQ ID NO: 60) CDPWVWYDLC; (SEQID NO: 61) CDPWIFFDRC; (SEQ ID NO: 62) CDPWMFFDQC; (SEQ ID NO: 63)CDPWLWYDRC; (SEQ ID NO: 64) CDVWVWYDQC; (SEQ ID NO: 65) CDPWIYYDLC; (SEQID NO: 66) CVPWTLFDLC; (SEQ ID NO: 67) CPAWYLEYMC; (SEQ ID NO: 68)CPDWYLEYMC; (SEQ ID NO: 69) CKYWQWFDKC; (SEQ ID NO: 70) and CDHWMWYDKC.(SEQ ID NO: 71)

Other preferred formula (I) sequences include, but are not limited tothe following: GCNREIEAMCCG; (SEQ ID NO: 72) GCPEWYTDVMCG; (SEQ ID NO:73) NWYCMDGEMAVDCEAT; (SEQ ID NO: 74) WQSCNMGWMSWPCYFV; (SEQ ID NO: 75)HELCETYADWLGCVEW; (SEQ ID NO: 76) PCDPWMFFDMCERW; (SEQ ID NO: 77)LRGCDPWIWYDLCPAV; (SEQ ID NO: 78) GYLCDPWIFYDRCLGF; (SEQ ID NO: 79)RFACDPWVFFDICGYW; (SEQ ID NO: 80) GYWCDPWTYYDLCLTA; (SEQ ID NO: 81)MWTCDPWIFYDRCFLN; (SEQ ID NO: 82) GSSCDPWLFYDLCLLD; (SEQ ID NO: 83)GGGCDPWVWYDLCWCD; (SEQ ID NO: 84) YTSCDPWIFFDRCMSV; (SEQ ID NO: 85)DPYCDPWMFFDQCAYL; (SEQ ID NO: 86) REFCDPWLWYDRCL; (SEQ ID NO: 87)NTGCDVWVWYDQCFAM; (SEQ ID NO: 88) LVFCDPWIYYDLCMDT; (SEQ ID NO: 89)GCSFVQLNSICG; (SEQ ID NO: 90) GCPAWYLEYMCG; (SEQ ID NO: 91)GCPDWYLEYMCG; (SEQ ID NO: 92) GCKYWQWFDKCG; (SEQ ID NO: 93) andGCDHWMWYDKCG. (SEQ ID NO: 94)

B. Compounds of formula (II):

In another aspect, compounds are provided comprising a peptide chainsequence of amino acids of formula (II) (II) X^(I) ₁X^(I) ₂X^(I)₃SGWVW^(I) ₄ (SEQ ID NO: 2)wherein each amino acid is indicated by the standard one-letterabbreviation, and wherein X^(I) ₁ is S, Q, R, L or Y; X^(I) ₂ is N, S,T, A or D; X^(I) ₃ is E, D or N; and X^(I) ₄ is L V, T, P or H.

Preferably X^(I) ₁ is S or Q; X^(I) ₂ is S; X^(I) ₃ is N; and X^(I) ₄ isV.

Examples of particularly preferred sequences satisfying formula (II)include, but are not limited to, the following: SNESGWVWL; (SEQ ID NO:95) QSNSGWVWV; (SEQ ID NO: 96) RTESGWVWT; (SEQ ID NO: 97) RANSGWVWV;(SEQ ID NO: 98) YDNSGWVWH; (SEQ ID NO: 99) and LSDSGWVWVP. (SEQ ID NO:100)

Other preferred formula (II) sequences include, but are not limited to,the following: EQSNSGWVWVGGGGC; (SEQ ID NO: 101) CEQSNSGWVWV; (SEQ IDNO: 102) EQSNSGWVWVGGGGCKKK; (SEQ ID NO: 103) EQSNSGWVWVGKKKC; (SEQ IDNO: 104) EQSNSGWVWVGKKK; (SEQ ID NO: 105) KKKEQSNSGWVWV; (SEQ ID NO:106) EQSNSGWVWVGKKKSKKK; (SEQ ID NO: 107) EQSNSGWVWVGGCKKK; (SEQ ID NO:108) EQSNSGWVWVGGGGGGCKKK; (SEQ ID NO: 109) SNESGWVWLP; (SEQ ID NO: 110)EQSNSGWVWV; (SEQ ID NO: 111) SRTESGWVWT; (SEQ ID NO: 112) QRANSGWVWV;(SEQ ID NO: 113) DYDNSGWVWH; (SEQ ID NO: 114) EQSNSGWVWVGKKKK; (SEQ IDNO: 115) EQSNSGWVWVGGGGSKKK; (SEQ ID NO: 116) EQSNSGWVWVGGGGS; (SEQ IDNO: 117) EQSNSGWVWVGGGGSEQSNSGWVWVGGGGS; (SEQ ID NO: 118)RYQSFELSDSGWVWVPVARH; (SEQ ID NO: 119) and EQSNSGWVWVGGGGCKKKC. (SEQ IDNO: 492)

C. Compounds of Formula (III):

In another aspect, the invention provides compounds comprising a peptidechain approximately 6 to 40 amino acids in length that binds to G-CSFRand contains a sequence of amino acids of formula (III) (III) ERX^(II)₁X^(II) ₂X^(II) ₃C (SEQ ID NO: 3)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(II) ₁ is D, L, S, G, E, A, K or Y; X^(II) ₂is W, Y, F, L or V; and X^(II) ₃ is F, G, M or L.

Preferably, X^(II) ₁ is D or L; X^(II) ₂ is W; and X^(II) ₃ is F.

Examples of particularly preferred sequences satisfying formula (III)include, but are not limited to, the following: ERDWFC; (SEQ ID NO: 120)ERDWGC; (SEQ ID NO: 121) ERLWFC; (SEQ ID NO: 122) ERSYFC; (SEQ ID NO:123) ERGWFC; (SEQ ID NO: 124) EREWFC; (SEQ ID NO: 125) ERAWFC; (SEQ IDNO: 126) ERLYFC; (SEQ ID NO: 127) ERYFMC; (SEQ ID NO: 128) ERLFLC; (SEQID NO: 129) ERALMC; (SEQ ID NO: 130) ERDVMC; (SEQ ID NO: 131) andERKWFC. (SEQ ID NO: 132)

Particulary preferred compounds are of the formula: ETWGERDWFC; (SEQ IDNO: 133) ETWGERDWGC; (SEQ ID NO: 134) STAERLWFCG; (SEQ ID NO: 135)YETAERSYFC; (SEQ ID NO: 136) ADNAERGWFC; (SEQ ID NO: 137) QSNSEREWFC;(SEQ ID NO: 138) STSERAWFCG; (SEQ ID NO: 139) ASWSERGWFC; (SEQ ID NO:140) ELSSEREWFC; (SEQ ID NO: 141) DMQGERGWFC; (SEQ ID NO: 142)SSSERAWFCG; (SEQ ID NO: 143) GNMRERLYFC; (SEQ ID NO: 144) QPNRERYFMC;(SEQ ID NO: 145) SVTRERLFLC; (SEQ ID NO: 146) IPLSERALMCSSWNC; (SEQ IDNO: 147) WARSERDVMCLSYVC; (SEQ ID NO: 148) QSNSEREWFCG; (SEQ ID NO: 149)QSNSEREWFCGGGGS; (SEQ ID NO: 150) NLEEALAQERLWFCRSGNC; (SEQ ID NO: 151)and NLESYEMEERKWFCKMFSC. (SEQ ID NO: 152)

D. Compounds of Formula (IV):

In another aspect, compounds are provided comprising a peptide chainapproximately 9 to 40 amino acids in length that binds to G-CSFR andcontains a sequence of amino acids of formula (IV): (IV) X^(III)₁MVYX^(III) ₂X^(III) ₃PX^(III) ₄W (SEQ ID NO: 4)wherein each amino acid in indicated by standard one-letterabbreviation, and wherein X^(III) ₁ is D or E; X^(III) ₂ is A or T;X^(III) ₃ is Y or V; and X^(III) ₄ is P or Y.

Examples of particularly preferred sequences satisfying formula (IV)include, but are not limited to, the following: DMVYAYPPW; (SEQ ID NO:153) and EMVYTVPYW. (SEQ ID NO: 154)

Other preferred formula (IV) sequences include, but are not limited to,the following: DMVYAYPPWS; (SEQ ID NO: 155) and DEMVYTVPYW. (SEQ ID NO:156)

E. Compounds of Formula (V):

In another aspect, compounds are provided comprising a peptide chainapproximately 12 to 40 amino acids in length that binds to G-CSFR andcontains a sequence of amino acids of formula (V): (V) CX^(IV) ₁X^(IV)₂X^(IV) ₃X^(IV) ₄X^(IV) ₅X^(IV) ₆X^(IV) ₇X^(IV) ₈X^(IV) ₉X^(IV) ₁₀C (SEQID NO: 5)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(IV) ₁ is E, G, P, N, R, T, W, S, L, H, A, Qor Y; X^(IV) ₂ is S, T, E, A, D, G, W, P, L, N, V, Y, R or M; X^(IV) ₃is R, Y, V, Q, E, T, L, P, S, K, M, A or W; X^(IV) ₄ is L, M, G, F, W,R, S, V, P, A, D, C or T; X^(IV) ₅ is V, T, A, R, S, L, W, C, I, E, P,H, F, D or Q; X^(IV) ₆ is E, Y, G, T, Q, M, S, N, A or P; X^(IV) ₇is C,V, D, G, L, W, E, V, I, S, M or A; X^(IV) ₈ is S, Y, A, W, P, V, L, Q,G, K, F, I, E or D; X^(IV) ₉ is R, W, M, D, H, V, G, A, Q, L, S, E or Y;X^(IV) ₁₀ is M, L, I, S, V, P, W, F, T, Y, R, or Q.

Preferably X^(IV) ₁ is E; X^(IV) ₂ is S or A; X^(IV) ₃ is R; X^(IV) ₄ isL; X^(IV) ₅ is V or S; X^(IV) ₆ is E; X^(IV) ₇ is C; X^(IV) ₈ is S;X^(IV) ₉ is R; and X^(IV) ₁₀ is L.

Examples of particularly preferred sequences satisfying formula (V)include, but are not limited to, the following: CESRLVECSRMC; (SEQ IDNO: 157) CETYMTYVYWLC; (SEQ ID NO: 158) CGERLAECARLC; (SEQ ID NO: 159)CESRLRECSMLC; (SEQ ID NO: 160) CEARLSECSRIC; (SEQ ID NO: 161)CPARLLECSRMC; (SEQ ID NO: 162) CESVGVGDWWSC; (SEQ ID NO: 163)CEDRLVEGPWVC; (SEQ ID NO: 164) CNDQFRTCVDVC; (SEQ ID NO: 165)CRGEWWELYHPC; (SEQ ID NO: 166) CEDTRTGWAWSC; (SEQ ID NO: 167)CTWLSSGELVWC; (SEQ ID NO: 168) CWPPVCEVSGIC; (SEQ ID NO: 169)CSLSPIQLQHLC; (SEQ ID NO: 170) CLARLEECSRFC; (SEQ ID NO: 171)CHNSSPMVGVTC; (SEQ ID NO: 172) CHVSPVQIKALC; (SEQ ID NO: 173)CAAPATSWFQYC; (SEQ ID NO: 174) CASKLHECSLRC; (SEQ ID NO: 175)CEPMDSNGIVQC; (SEQ ID NO: 176) CQYASAADEQRC; (SEQ ID NO: 177)CEYWDEPSLSWC; (SEQ ID NO: 178) CERECFQMLERC; (SEQ ID NO: 179)CGMSTDELDEIC; (SEQ ID NO: 180) CYVSPSTGLYSC; (SEQ ID NO: 181)CEARLVECSRLC; (SEQ ID NO: 182) CESRLSECSRMC; (SEQ ID NO: 183)CELKLQECARRC; (SEQ ID NO: 184) CELKLQEAARRC; (SEQ ID NO: 185) andCLERLEECSRFC. (SEQ ID NO: 186)

Other preferred formula (V) sequences include but are not limited to,the following: GGCESRLVECSRMC; (SEQ ID NO: 187) GGCETYMTYVYWLC; (SEQ IDNO: 188) EWLCESVGVGDWWSC; (SEQ ID NO: 189) YHPCEDRLVEGPWVCCRS; (SEQ IDNO: 190) WLLCNDQFRTCVDVCDNV; (SEQ ID NO: 191) IAECRGEWWELYHPCLAA; (SEQID NO: 192) TWYCEDTRTGWAWSCLEL; (SEQ ID NO: 193) QLDCTWLSSGELVWCSDW;(SEQ ID NO: 194) QFDCTWLSSGELVWCSDW; (SEQ ID NO: 195) CWPPVCEVSGICS;(SEQ ID NO: 196) CGCSLSPIQLQHLC; (SEQ ID NO: 197) CGCHVSPVQIKALC; (SEQID NO: 198) GCHVSPVQIKALC; (SEQ ID NO: 199) GTSCAAPATSWFQYCVLP; (SEQ IDNO: 200) RMDCASKLHECSLRCAYA; (SEQ ID NO: 201) GVVCEPMDSNGIVQCSMR; (SEQID NO: 202) IDVCQYASAADEQRCLRI; (SEQ ID NO: 203) NVLCEYWDEPSLSWCLSS;(SEQ ID NO: 204) CQCERECFQMLERC; (SEQ ID NO: 205) FCSCGMSTDELDEICAIW;(SEQ ID NO: 206) EEVCYVSPSTGLYSCYDQ; (SEQ ID NO: 207) LLDICELKLQECARRCN;(SEQ ID NO: 208) GGGLLDICELKLQECARRCN; (SEQ ID NO: 209)GRTGGGLLDICELKLQECARRCN; (SEQ ID NO: 210) LGIEGRTGGGLLDICELKLQECARRCN;(SEQ ID NO: 211) LLDICELKLQEAARRCN; (SEQ ID NO: 212) andKLLDICELKLQEAARRCN. (SEQ ID NO: 213)

Particularly preferred formula (V) sequences are selected from the groupconsisting of: LLDICELKLQECARRCN; (SEQ ID NO: 208) GGGLLDICELKLQECARRCN;(SEQ ID NO: 209) GRTGGGLLDICELKLQECARRCN; (SEQ ID NO: 210)LGIEGRTGGGLLDICELKLQECARRCN; (SEQ ID NO: 211) LLDICELKLQEAARRCN; (SEQ IDNO: 212) and KLLDICELKLQEAARRCN. (SEQ ID NO: 213)

F. Compounds of Formula (VI):

In another aspect, compounds are provided comprising a peptide chainapproximately 9 to 40 amino acids in length that binds to G-CSFR andcontains a sequence of amino acids of formula (VI): (VI) X^(V) ₁X^(V)₂X^(V) ₃X^(V) ₄X^(V) ₅X^(V) ₆CX^(V) ₇X^(V) ₈ (SEQ ID NO: 6)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(V) ₁ is E, C, Q, V, or Y; X^(V) ₂ is E, A,L, M, S, W, or Q; X^(V) ₃ is K, R or T; X^(V) ₄ is L, A, or V; X^(V) ₅is R, A, M, H, E, V, L, G, D, Q, or S; X^(V) ₆ is E or V; X^(V) ₇ is Aor G; X^(V) ₈ is R, H, G or L.

Preferably X^(V) ₁ is E; X^(V) ₂ is A or L; X^(V) ₃ is K or R; X^(v) ₄is L; X^(V) ₆ is E; X^(V) ₇ is A; and X^(V) ₈ is R.

Examples of particularly preferred sequences satisfying formula (VI)include, but are not limited to, the following: EEKLRECAR; (SEQ ID NO:214) EARLAECAR; (SEQ ID NO: 215) CMKLMECAR; (SEQ ID NO: 216) ELRLRECAH;(SEQ ID NO: 217) EAKLHECAR; (SEQ ID NO: 218) ELKLAECAR; (SEQ ID NO: 219)EARLEECAR; (SEQ ID NO: 220) EAKLRECAR; (SEQ ID NO: 221) ELRLAECAR; (SEQID NO: 222) ESRLAECAR; (SEQ ID NO: 223) EAKLVECAR; (SEQ ID NO: 224)ESRLRECAR; (SEQ ID NO: 225) EAKLAECAR; (SEQ ID NO: 226) QWRLEECAR; (SEQID NO: 227) QLRLEECAR; (SEQ ID NO: 228) ELRLEECAR; (SEQ ID NO: 229)EAKLLECAR; (SEQ ID NO: 230) EARAGVCAG; (SEQ ID NO: 231) EAKAGVCAG; (SEQID NO: 232) VARLEECAR; (SEQ ID NO: 233) ELKLDECAR; (SEQ ID NO: 234)EWRLQECAR; (SEQ ID NO: 235) EAKLSECAR; (SEQ ID NO: 236) EARLSECAR; (SEQID NO: 237) ELKLLECAR; (SEQ ID NO: 238) ELRLQECGR; (SEQ ID NO: 239)EQKLAECAR; (SEQ ID NO: 240) ELRLQECAR; (SEQ ID NO: 241) ELKLEECAR; (SEQID NO: 242) ESRLEECAR; (SEQ ID NO: 243) EATVQECAR; (SEQ ID NO: 244)ELKLQECAR; (SEQ ID NO: 245) YSRLEECGR; (SEQ ID NO: 246) ELRLRECAL; (SEQID NO: 247) EARLLECAR; (SEQ ID NO: 248) ESRLLECAR; (SEQ ID NO: 249)VLKLEECAR; (SEQ ID NO: 250) ESKLAECAR; (SEQ ID NO: 251) ESKLRECAR; (SEQID NO: 252) EYKLGECAR; (SEQ ID NO: 253) ESRLQECAR; (SEQ ID NO: 254)QARLAECAR; (SEQ ID NO: 255) ELKKQECAR; (SEQ ID NO: 256) ESRLSECAR; (SEQID NO: 257) EARLEECGR; (SEQ ID NO: 258) ESRLAECGR; (SEQ ID NO: 259)EWRLEECAR; (SEQ ID NO: 260) EARLSECGR; (SEQ ID NO: 261) AARLAECAR; (SEQID NO: 262) EWKLAECAR; (SEQ ID NO: 263) ESKLEECAR; (SEQ ID NO: 264)DVKLAECAR; (SEQ ID NO: 265) ELQLEECAR; (SEQ ID NO: 266) and EYKLASCAR.(SEQ ID NO: 267)

Other preferred formula (VI) sequences include but are not limited to,the following: RLSICEEKLRECARGC; (SEQ ID NO: 268) PLTTCEARLAECARQL; (SEQID NO: 269) LALCMKLMECARRY; (SEQ ID NO: 270) ELVMCELRLRECAHRA; (SEQ IDNO: 271) PLARCEAKLHECARQL; (SEQ ID NO: 272) LLSVCELKLAECARSK; (SEQ IDNO: 273) RLEWCEARLEECARRC; (SEQ ID NO: 274) RLRVVEAKLRECARGR; (SEQ IDNO: 275) CVAHLELRLAECARQI; (SEQ ID NO: 276) HLARCESRLAECARQL; (SEQ IDNO: 277) RLALLEAKLVECARRL; (SEQ ID NO: 278) DLFSLESRLRECARRV; (SEQ IDNO: 279) AVPVLEAKLAECARRF; (SEQ ID NO: 280) YLQQLQWRLEECARGM; (SEQ IDNO: 281) YLELCQLRLEECARQFN; (SEQ ID NO: 282) ELHICELRLEECARGR; (SEQ IDNO: 283) RVARCELRLAECARKS; (SEQ ID NO: 284) YLEVLESRLAECARWK; (SEQ IDNO: 285) EAKLLECARAR; (SEQ ID NO: 286) ELSLCEARAGVCAGSVTK; (SEQ ID NO:287) ELSLCEAKAGVCAGSVTK; (SEQ ID NO: 288) ALWQCVARLEECARSR; (SEQ ID NO:289) CLKSCELKLDECARRM; (SEQ ID NO: 290) ALQTCEWRLQECARSR; (SEQ ID NO:291) YISQCEAKLAECARLY; (SEQ ID NO: 292) ELSSCEAKLSECARRW; (SEQ ID NO:293) ELSSCEARLSECARRW; (SEQ ID NO: 294) QLLQCELKLLECARQG; (SEQ ID NO:295) ELLRCEARLAECARGC; (SEQ ID NO: 296) QLRQCELRLQECGRHGN; (SEQ ID NO:297) PLTSCEQKLAECARRF; (SEQ ID NO: 298) LLGMCELRLQECARAK; (SEQ ID NO:299) ELSRCELKLEECARGM; (SEQ ID NO: 300) DCRPCESRLEECARRL; (SEQ ID NO:301) RLSVCEARLEECARQL; (SEQ ID NO: 302) PLKMCEATVQECARLI; (SEQ ID NO:303) LLLFCEARLSECARHV; (SEQ ID NO: 304) SLSMCEARLAECARLL; (SEQ ID NO:305) PLFSCELKLQECARRCN; (SEQ ID NO: 306) SLERCYSRLEECGRRI; (SEQ ID NO:307) PLTSCELRLRECALRSN; (SEQ ID NO: 308) KLAACELKLAECARRW; (SEQ ID NO:309) KLAACELRLAECARRW; (SEQ ID NO: 310) ALTRCELRLAECARKI; (SEQ ID NO:311) LLQQCELKLAECARSI; (SEQ ID NO: 312) QLWQCEARLLECARRS; (SEQ ID NO:313) RLRLCESRLLECARSL; (SEQ ID NO: 314) QLETCVLKLEECARRCN; (SEQ ID NO:315) ALSQCELRLAECARSVTK; (SEQ ID NO: 316) ELKLAECARRS; (SEQ ID NO: 317)ALSRCESKLAECARRQ; (SEQ ID NO: 318) LMSTCESKLRECARSL; (SEQ ID NO: 319)SLQRCEYKLGECARSL; (SEQ ID NO: 320) RLELLESRLQECARQLN; (SEQ ID NO: 321)QMEWCQARLAECARCCN; (SEQ ID NO: 322) PLFSCELKKQECARRCN; (SEQ ID NO: 323)LLDKCESRLSECARRL; (SEQ ID NO: 324) LLARCEARLEECGRQC; (SEQ ID NO: 325)DLLYCESRLAECGRM; (SEQ ID NO: 326) ALQMCEWRLEECARRL; (SEQ ID NO: 327)LLTMCEARLSECGRRL; (SEQ ID NO: 328) ALWRCESRLAECARRS; (SEQ ID NO: 329)LLATCAARLAECARQL; (SEQ ID NO: 330) LQTCEWKLAECARSN; (SEQ ID NO: 331)PLRSCESKLEECARQL; (SEQ ID NO: 332) CLRALDVKLAECARHL; (SEQ ID NO: 333)RLKTLELQLEECARRS; (SEQ ID NO: 334) KLRDVELKLAECARRS; (SEQ ID NO: 335)SLQRCEYKLASCARSL; (SEQ ID NO: 336) RLARCELRLAECARKS; (SEQ ID NO: 337)DLWYLESKLEECARRCN; (SEQ ID NO: 338) DLWYLESKLEECARRANG; (SEQ ID NO: 339)DLWYLESKLEECARRCNG; (SEQ ID NO: 340) KQRELELKLAECARRS; (SEQ ID NO: 341)QMQEWCARLAECARCCN; (SEQ ID NO: 342) and LLDICELKLQECARRAN. (SEQ ID NO:343)

A particularly preferred sequence of formula (VI) is: LLDICELKLQECARRAN.(SEQ ID NO: 343)

G. Compounds of Formula (VII):

In another aspect, the invention provides compounds comprising a peptidechain approximately 10 to 40 amino acids in length that binds to G-CSFRand contains a sequence of amino acids of formula (VII): (VII) X^(VI)₁X^(VI) ₂X^(VI) ₃X^(VI) ₄X^(VI) ₅EX^(VI) ₆X^(VI) ₇X^(VI) ₈X^(VI) ₉ (SEQID NO: 7)wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(VI) ₁ is A, E or G; X^(VI) ₂ is E, H or D;X^(VI) ₃ is R or G; X^(VI) ₄ is K, Y, M, N, Q, R, D, I, S or E; X^(VI) ₅is A, S or P; X^(VI) ₆ is E, D, T, Q, K or A: X^(VI) ₇ is R, W, K, L, S,A or Q; X^(VI) ₈ is R or E; and X^(VI) ₉ is W, G, or R.

Preferably X^(VI) is A; X^(VI) ₂ is E; X^(VI) ₃ is R; X^(VI) ₅ is A;X^(VI) ₆ is E; X^(VI) ₇ is R; X^(VI) ₈ is R or E; and R; and X^(VI) ₉ isW.

Examples of particularly preferred sequences satisfying formula (VII)include, but are not limited to, the following: AERKAEERRW; (SEQ ID NO:344) AERYAEEREG; (SEQ ID NO: 345) AERMAEERRW; (SEQ ID NO: 346)AERKAEERRR; (SEQ ID NO: 347) AHRNAEERRW; (SEQ ID NO: 348) AERKSEDWRW;(SEQ ID NO: 349) AERKAEEKRR; (SEQ ID NO: 350) AERQAETRRW; (SEQ ID NO:351) AERNAEERRW; (SEQ ID NO: 352) AERQAEERRW; (SEQ ID NO: 353)AERRAEERRW; (SEQ ID NO: 354) AERDAEQRRW; (SEQ ID NO: 355) AERIAEERRW;(SEQ ID NO: 356) AERSAEERRW; (SEQ ID NO: 357) AERKAEELRW; (SEQ ID NO:358) AERKAEESRW; (SEQ ID NO: 359) EERKAEERRW; (SEQ ID NO: 360)ADGKAEERRW; (SEQ ID NO: 361) ADGKAEELRW; (SEQ ID NO: 362) ADGMPEERRW;(SEQ ID NO: 363) ADGEAEKRRW; (SEQ ID NO: 364) ADGNAEERRW; (SEQ ID NO:365) ADGEAEKARW; (SEQ ID NO: 366) AEGEAEKARW; (SEQ ID NO: 367)GERKAEERRW; (SEQ ID NO: 368) AEREAEERRW; (SEQ ID NO: 369) ADGEAEARRW;(SEQ ID NO: 370) ADGRAEEARW; (SEQ ID NO: 371) AEGRAEEARW; (SEQ ID NO:372) AEREAEKARW; (SEQ ID NO: 373) AERKAEEQRW; (SEQ ID NO: 374)AERDAEKRRW; (SEQ ID NO: 375) and AEREAEKLRW. (SEQ ID NO: 376)

Other preferred formula (VI) sequences include but are not limited to,the following: MLAERKAEERRWFNTHGRE; (SEQ ID NO: 377)MLAERKAEERRWFNTHGREK; (SEQ ID NO: 378) GGGMLAERKAEERRWFNTHGRE; (SEQ IDNO: 379) CMLAERKAEERRWFNTHGRE; (SEQ ID NO: 380) CMLAERKAEERRWFNTHGREK;(SEQ ID NO: 381) MLAERYAEEREGFNMQWRE; (SEQ ID NO: 382)MLAERMAEERRWFRRMG; (SEQ ID NO: 383) IVAERKAEERRRLNTEGHE; (SEQ ID NO:384) ILAHRNAEERRWFQKHGR; (SEQ ID NO: 385) MLAERKSEDWRWLKTHGRD; (SEQ IDNO: 386) MLAERKAEEKRRLKTQGRE; (SEQ ID NO: 387) ILAERQAETRRWMRNAGSVTK;(SEQ ID NO: 388) MLAERNAEERRWLKRQCG; (SEQ ID NO: 389)MLAERQAEERRWLKMHGGE; (SEQ ID NO: 390) MLAERRAEERRWLKTQGGD; (SEQ ID NO:391) MLAERQAEERRWLKTQGRD; (SEQ ID NO: 392) MLAERKAEERRWFKTHGRE; (SEQ IDNO: 393) MLAERKAEERRWFNNQGRE; (SEQ ID NO: 394) MPAERDAEQRRWLKTHGRE; (SEQID NO: 395) ILAERIAEERRWLKTQGR; (SEQ ID NO: 396) MLAERKAEERRWLQTHGRE;(SEQ ID NO: 397) ILAERSAEERRWLKTQGRE; (SEQ ID NO: 398)LLAERKAEELRWLKTHGRE; (SEQ ID NO: 399) MLAERKAEERRWLQTHGRE; (SEQ ID NO:400) MLAERNAEERRW; (SEQ ID NO: 401) MFAERKAEESRWLQSQGRE; (SEQ ID NO:402) MLEERKAEERRWLKTHGR; (SEQ ID NO: 403) MLAERKAEERRWLKMQGRE; (SEQ IDNO: 404) MLAERNAEERRWFYTHGRE; (SEQ ID NO: 405) MLADGKAEERRWLKTHGLD; (SEQID NO: 406) MIADGKAEERRWLKTHGRD; (SEQ ID NO: 407) MLADGKAEELRWLKTQGSD;(SEQ ID NO: 408) MLAERNAEERRWLKTHGRD; (SEQ ID NO: 409)MLADGKAEELRWLKTQGRE; (SEQ ID NO: 410) ILADGKAEERRWLKTHGRD; (SEQ ID NO:411) MLADGMPEERRWLQTHGRD; (SEQ ID NO: 412) MLADGEAEKRRWLNTHGRD; (SEQ IDNO: 413) MLADGNAEERRWLMTHGRD; (SEQ ID NO: 414) MLADGEAEKARWLKTQGRE; (SEQID NO: 415) MLAEGEAEKARWLKTQGRE; (SEQ ID NO: 416) MLADGKAEERRWLKTQGRE;(SEQ ID NO: 417) MLAERKAEERRWLSAHVRE; (SEQ ID NO: 418)LLGERKAEERRWYKTHARE; (SEQ ID NO: 419) MLAERKAEERRWLMTHGHD; (SEQ ID NO:420) MLAERKAEERRWLKSQCLE; (SEQ ID NO: 421) LLAEREAEERRWFKTHGRE; (SEQ IDNO: 422) MLADGEAEARRWFNMHGRE; (SEQ ID NO: 423) MLADGRAEEARWLKTQGSE; (SEQID NO: 424) MLAEGRAEEARWLKTQGSE; (SEQ ID NO: 425) MLAEREAEKARWLKTQGRE;(SEQ ID NO: 426) MMAERKAEEQRWFDIHGRD; (SEQ ID NO: 427)LTAERDAEKRRWLLTHGGE; (SEQ ID NO: 428) MLAERQAEERRWLKSQRGE; (SEQ ID NO:429) LLAERKAEERRWFATHGRD; (SEQ ID NO: 430) MLAEREAEKLRWLKSQERA; (SEQ IDNO: 431) MLAERKAEERRWLKTHGGE; (SEQ ID NO: 432) KGGGMLAERKAEERRWFNTHGRE;(SEQ ID NO: 490) and KSTGGLTAERDAEKRRWLLTHGGE. (SEQ ID NO: 491)

H. Other Active Compounds

In another aspect of the invention, there are provided additionalcompounds comprising a peptide chain approximately 5 to 40 amino acidsin length that binds to G-CSFR and contains a sequence of amino acidsselected from the following compounds: CTWTDLESVY; (SEQ ID NO: 433)HTTNEQFFMC; (SEQ ID NO: 434) DTWLELESRY; (SEQ ID NO: 435) HNSSPMVGVT;(SEQ ID NO: 436) DWQKTIPAYW; (SEQ ID NO: 437) RWGREGLVAALL; (SEQ ID NO:438) WSGTRVWRCVVT; (SEQ ID NO: 439) MSLLSYLRS; (SEQ ID NO: 440) LDLLAI;(SEQ ID NO: 441) RIYGVK; (SEQ ID NO: 442) MIWHMFMSLLF; (SEQ ID NO: 443)FFWASWMHLLW; (SEQ ID NO: 444) FDDCWREREQFLFQAL; (SEQ ID NO: 445)CGRASECFRLLEM; (SEQ ID NO: 446) RECFQMLER; (SEQ ID NO: 447)CSIRWDFVPGYGLC; (SEQ ID NO: 448) WMQCWDSLSLCYDM; (SEQ ID NO: 449)ALLMCESKLAECARAR; (SEQ ID NO: 450) LAHCKKRKEECAAG; (SEQ ID NO: 451)SIDGVYLRTSRT; (SEQ ID NO: 452) SIDGVYLRTRSRTRY; (SEQ ID NO: 453)VRWLRGSTLRGLRDR; (SEQ ID NO: 454) DRGGGTVGVYWWESY; (SEQ ID NO: 455)VWGTVGTWLEY; (SEQ ID NO: 456) LMWVSAY; (SEQ ID NO: 457)RASDEYGALVRFCTNL; (SEQ ID NO: 458) NYWCDSNWVCEIA; (SEQ ID NO: 459)LAHCLLRLEECAAG; (SEQ ID NO: 460) LALCLARLRECAGG; (SEQ ID NO: 461)CESRLVECSRM; (SEQ ID NO: 462) LLDIAELKLQECARRCN; (SEQ ID NO: 463)KLLDIAELKLQECCARRCN; (SEQ ID NO: 464) CSTGGGLTAERDAEKRRWLLTHGGE (SEQ IDNO: 465) LTAERDAEKRRWLLTHGGEGG; (SEQ ID NO: 466) LTAERDAEKRRWLLTHGGEGGK;(SEQ ID NO: 467) LTAERDAEKRRWLLTHGGEGGGGG; (SEQ ID NO: 468)LTAERDAEKRRWLLTHGGEGGGGGK; (SEQ ID NO: 469) ESGWVW; (SEQ ID NO: 470)NSGWVW; (SEQ ID NO: 471) SGWVW; (SEQ ID NO: 472) PLGKCEATCREMARYFN; (SEQID NO: 473) SLQRCEYKLASVRGLCN (SEQ ID NO: 474) DLWYLESKLEEAARRCNG; (SEQID NO: 475) PYMGTRSRAKLLRQQ; (SEQ ID NO: 476) RNAGERRWFKTQGWY; (SEQ IDNO: 477) MLAERNADDRRWFNTHGRD; (SEQ ID NO: 478) MMADGRLRNSVGLILWCD; (SEQID NO: 479) MLADGRLRNVVG; (SEQ ID NO: 480) LLADVRRRNGVGLLRMGRD; (SEQ IDNO: 481) MLADGRLRNFGG; (SEQ ID NO: 482) TYMTYVYWLC; (SEQ ID NO: 483)RFGERWGL; (SEQ ID NO: 484) HWLWWGWNF; (SEQ ID NO: 485) RECFQMLERC; (SEQID NO: 486) ILAHRNAKERRWFQKHGR; (SEQ ID NO: 487) andCSTGGGLTAERDAEKRRWLLTHGGEK. (SEQ ID NO: 489)

Particularly preferred sequences are selected from the group consistingof: LLDIAELKLQECARRCN; (SEQ ID NO: 463) and KLLDIAELKLQECCARRCN. (SEQ IDNO: 464)

I. Synthesis of the Peptides:

Standard solid phase peptide synthesis techniques are preferred forsynthesis of the peptides of the present invention. Such techniques aredescribed, for example, by Merrifield (1963) J. Chem. Soc. 85:2149. Asis well known in the art, solid phase synthesis using the Merrifieldmethod involves successive coupling of α-amino protected amino acids toa growing support-bound peptide chain. After the initial coupling of aprotected amino acid to a resin support (e.g., a polystyrene resin, achloromethylated resin, a hydroxymethyl resin, a benzhydrylamine resin,or the like, depending on the chemistry used), the α-amino protectinggroup is removed by a choice of reagents, depending on the specificprotecting group. Suitable α-amino protecting groups are those known tobe useful in the art of stepwise synthesis of peptides. Included areacyl type protecting groups (e.g., formyl, trifluoroacetyl, acetyl),aromatic urethane type protecting groups (e.g., benzyloxycarbonyl (Cbz)and substituted Cbz), aliphatic urethane protecting groups (e.g.,t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl),alkyl type protecting groups (e.g., benzyl, triphenylmethyl),fluorenylmethyl oxycarbonyl (Fmoc), alloxycarbonyl (Alloc) and Dde. Theside chain protecting groups (typically ethers, esters, trityl, and thelike) remain intact during coupling; however, the side chain protectinggroup must be removable upon completion of the synthesis of the finalpeptide. Preferred side chain protecting groups, as will appreciated bythose skilled in the art, will depend on the particular amino acid thatis being protected as well as the overall chemistry used. After removalof the α-amino protecting group, the remaining protected amino acids arecoupled stepwise in the desired order. Each protected amino acid isgenerally reacted in about a 3-fold excess using an appropriate carboxylgroup activator such as 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) ordicyclohexylcarbodiimide (DCC) in solution, for example, in methylenecloride (CH₂Cl₂), N-methyl pyrrolidone, dimethyl formamide (DMF), ormixtures thereof.

Once the synthesis is complete, the compound is cleaved from the solidsupport by treatment with a reagent such as trifluoroacetic acid,preferably in combination with a scavenger such as ethanedithiol,β-mercaptoethanol or thioanisole. The cleavage reagent not only cleavesthe peptide from the resin, but also cleaves all remaining side chainprotecting groups.

These procedures can also be used to synthesize peptides containingamino acids other than the 20 naturally occurring, genetically encodedamino acids. For instance, naphthylalanine can be substituted fortryptophan, with 1-Nal or 2-Nal. Other synthetic amino acids that can besubstituted into the peptides of the present invention include, but arenot limited to, nor-leucine and 3-pyridylalanine.

III. Variation and Modification of the Compounds

A. Dimer Forms (with a Terminal Linking Moiety):

The compounds of the present invention may be in the form of a dimer,i.e., a compound comprised of two similar (but not necessarilyidentical) peptide sequences. Preferably, the dimer compounds of theinvention have the structure of formula (VIII)

wherein R¹, R², n1, n2, n3, n4, x, y and Lk are defined as follows.

R¹ is a peptide chain that binds to G-CSFR and contains a sequence ofamino acids of the present invention. R² is also a peptide chain thatbinds to G-CSFR and contains a sequence of amino acids of the presentinvention. As previously indicated, R¹ and R² can be the same ordifferent. It is preferred, however, that R¹ and R² are the same.

βA is a β-alanine residue and may or may not be present, meaning thatn1, n2, n3 and n4 are independently zero or 1.

Lk is a terminal linking moiety. If the dimer contains only one linkingmoiety, one of x and y is zero and the other is one. Alternatively, ifthe dimer contains two linking moieties, both x and y are one. Thus, xand y are independently zero or one with the proviso that the sum of xand y is either one or two.

The terminal linking moiety Lk can be any moiety recognized by thoseskilled in the art as suitable for joining the peptides of R¹ and R². Lkis preferably although not necessarily selected from the groupconsisting of a disulfide bond, a carbonyl moiety and a C₁₋₁₂ linkingmoiety optionally terminated with one or two —NH— linkages andoptionally substituted at one or more available carbon atoms with alower alkyl substituent. Preferably, the terminal linking moietycomprises —NH—R³—NH— wherein R³ is lower (C₁₋₆) alkylene substitutedwith a functional group such as a carboxyl group or an amino group thatenables coupling to another molecular moiety (e.g., as may be present onthe surface of a solid support), and is optionally substituted with alower alkyl group. Optimally, the linking moiety is a lysine residue orlysine amide, i.e., a lysine residue wherein the carboxyl group has beenconverted to an amide moiety —CONH₂.

(SEQ ID NO: 101)  (SEQ ID NO: 101);

(SEQ ID NO: 465)  (SEQ ID NO: 489);

(SEQ ID NO: 377)  (SEQ ID NO: 378);

(SEQ ID NO: 380)  (SEQ ID NO: 381);

(SEQ ID NO: 466)  (SEQ ID NO: 467); and

(SEQ ID NO: 468)  (SEQ ID NO: 469).

B. Disulfide Bonds:

When a pair of cysteine residues is present in a peptide of theinvention, it is preferred that the pair form a disulfide bond linkingthese residues. The disulfide bond may be present within a singlepeptide chain forming an intramolecular disulfide bond. Alternatively,if the compound includes an additional cysteine-containing peptidechain, the disulfide bond may connect the two chains. In addition, wherean additional pair of cysteine residues exists in the compound, morethan one disulfide bond may be present.

Disulfide bond formation may be effected by techniques well known tothose skilled in the art. One such technique involves employing asuitable oxidizing reagent such that a disulfide bond forms from thefree thiols from a pair of cysteine residues. Undesired disulfide bondformation can be minimized, for example, by protecting the thiol groupsof those cysteine residues not intended to form disulfide bonds andoxidizing the peptide before removal of any protecting groups. Preferredcompounds having disulfide bonds include, by way of example, thefollowing:

(SEQ ID NO: 135)  (SEQ ID NO: 135);

(SEQ ID NO: 138)  (SEQ ID NO: 138);

(SEQ ID NO: 149)  (SEQ ID NO: 149);

(SEQ ID NO: 339)  (SEQ ID NO: 339);

(SEQ ID NO: 475)  (SEQ ID NO: 475);

(SEQ ID NO: 340);

(SEQ ID NO: 343);

(SEQ ID NO: 212);

(SEQ ID NO: 231);

(SEQ ID NO: 463);

(SEQ ID NO: 464); and

(SEQ ID NO: 208)  (SEQ ID NO: 208).

A particularly preferred compound having disulfide bonds includes NH₃⁺-LLDICELKLQECARRCN-COO (SEQ ID NO: 208)           |      |   | NH₃⁺-LLDICELKLQECARRCN-COO. (SEQ ID NO: 208)

C. N-Terminal Modifications:

(i) PEGylated Compounds

The peptides and compounds of the invention can advantageously bemodified with or covalently coupled to one or more of a variety ofhydrophilic polymers. It has been found that when the peptide compoundsare derivatized with a hydrophilic polymer, their solubility andcirculation half-lives are increased and their immunogenicity is masked.Quite surprisingly, the foregoing can be accomplished with little, ifany, diminishment in binding activity. Nonproteinaceous polymerssuitable for use in accordance with the present invention include, butare not limited to, polyalkylethers as exemplified by polyethyleneglycol and polypropylene glycol, polylactic acid, polyglycolic acid,polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose andcellulose derivatives, dextran and dextran derivatives, etc. Generally,such hydrophilic polymers have an average molecular weight ranging fromabout 500 to about 100,000 daltons, more preferably from about 2,000 toabout 60,000 daltons and, even more preferably, from about 5,000 toabout 50,000 daltons. In preferred embodiments, such hydrophilicpolymers have average molecular weights of about 5,000 daltons, 10,000daltons 20,000 daltons and 40,000 daltons.

The peptide compounds of the invention can be derivatized with orcoupled to such polymers using any of the methods set forth in Zallipsky(1995) Bioconjugate Chem. 6:150-165; Monfardini et al. (1995)Bioconjugate Chem. 6:62-69; U.S. Pat. No. 4,640,835; U.S. Pat. No.4,496,689; U.S. Pat. No. 4,301,144; U.S. Pat. No. 4,670,417; U.S. Pat.No. 4,791,192; U.S. Pat. No. 4,179,337 or WO 95/34326.

In a preferred embodiment, the N-terminus of a peptide of the inventionis coupled to a polyethylene glycol molecule. It is particularlypreferred that the polymer is selected from the group consistingof:polyethylene glycol, polypropylene glycol, polylactic acid,polyglycolic acid and derivatives and combinations-thereof. Mostpreferably the polymer is polyethylene glycol (PEG), in which case thepeptide is referred to as “PEGylated.” PEG is a linear, water-solublepolymer of ethylene oxide repeating units with two terminal hydroxylgroups. PEGs are classified by their molecular weights which typicallyrange from about 500 daltons to about 40,000 daltons. In a presentlypreferred embodiment, the PEGs employed have an average molecular weightof from about 500 to about 80,000 daltons. It is particularly preferredthat the polymer has an average molecular weight of between about 5,000to 40,000 daltons.

The PEG coupled to the peptide compounds of the invention can be eitherbranched or unbranched. (See, e.g. Monfardini et al. (1995) BioconjugateChem. 6:62-69.) PEG is commercially available from Shearwater Polymers,Inc. (Huntsville, Ala.), Sigma Chemical Co. and other companies.Suitable PEGs include, but are not limited to, monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S—NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES) andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

Briefly, in one exemplary embodiment, the hydrophilic polymer which isemployed, e.g., PEG, is capped at one terminus by an unreactive groupsuch as a methoxy or ethoxy group. Thereafter, the polymer is activatedat the other terminus by reaction with a suitable activating agent, suchas a cyanuric halide (e.g., cyanuric chloride, bromide or fluoride),diimidazole, an anhydride reagent (e.g., a dihalosuccinic anhydride,such as dibromosuccinic anhydride), acyl azide, p-diazoniumbenzyl ether,3-(p-diazoniumphenoxy)-2-hydroxypropylether, or the like. The activatedpolymer is then reacted with a peptide compound of the invention toproduce a polymer-derivatized peptide compound. Alternatively, afunctional group in the peptide compounds of the invention can beactivated for reaction with the polymer, or two groups can be joined ina concerted coupling reaction using known coupling methods. It will bereadily appreciated that the peptide compounds of the invention can bederivatized with PEG using a myriad of other reaction schemes known tothose of skill in the art.

(ii) Acetylated Compounds

In some instances, the N-terminus of the peptide is acetylated.Preferred acetylated compounds include, by way of example, thefollowing: Ac-ESGWVW-CONH₂; (SEQ ID NO: 470) Ac-NSGWVW-CONH₂; (SEQ IDNO: 471) and Ac-SGWVW-CONH₂. (SEQ ID NO: 472)

The peptides and compounds of the invention can be modified with anacetyl moiety (Ac) using standard techniques known to those skilled inthe art. One such technique includes combining the peptide with anacetylating reagent (e.g., acetyl chloride, acetic anhydride) in asuitable solvent to form the acetylated product. To the extent thatother acetylated products are formed during the reaction, the N-terminusderivative can be isolated using conventional separation techniques.

D. C-Terminal Modifications:

The peptides and compounds of the invention can advantageously bemodified to include an amide functionality at the carboxyl terminus ofthe peptide. Thus, it is preferred that the C-terminus of the peptide isamidated.

In preparing peptides wherein the C-terminus carboxyl group is replacedby the amide —C(O)NR³R⁴ where R³ and R⁴ are independently H or lower(C₁₋₆)alkyl, a benzhydrylamine resin is preferably used as the solidsupport for peptide synthesis. Upon completion of the synthesis, ahydrogen fluoride treatment is employed to release the peptide from thesupport, directly resulting in the free peptide amide (i.e., theC-terminus is —C(O)NH₂). Alternatively, use of a chloromethylated resinduring peptide synthesis coupled with reaction with ammonia (to cleavethe side chain protected peptide from the support) yields the freepeptide amide and reaction with an alkylamine or a dialkylamine yields aside chain protected alkylamide or dialkylamide (i.e., the C-terminus is—C(O)NR³R⁴ where R³ and R⁴ are as defined above). Side chain protectinggroups are then removed in the usual fashion by treatment with hydrogenfluoride to give the free amides, alkylamides, or dialkylamides.

E. Other Modifications:

One can also replace the naturally occurring side chains of the 20genetically encoded amino acids (or the stereoisomeric D amino acids)with other side chains, for instance with groups such as alkyl, loweralkyl, cyclic 4-, 5-, 6- or 7-membered alkyl, amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lowerester derivatives thereof, and 4-, 5-, 6- or 7-membered heterocyclic. Inparticular, proline analogues in which the ring size of the prolineresidue is changed from 5 members to 4, 6, or 7 members can be employed.

One can also readily modify the peptides herein by phosphorylation orother methods as described in Hruby et al. (1990) Biochem J.268:249-262. Thus, the peptides of the invention also serve asstructural models for non-peptide compounds with similar biologicalactivity. For example, the peptide backbones may be replaced with abackbone composed of phosphonates, amidates, carbamates, sulfonamides,secondary amines, and N-methylamino acids.

IV. Utility

The compounds of the invention are useful in vitro as unique tools forunderstanding the biological role of G-CSF, including the evaluation ofthe many factors thought to influence, and be influenced by, theproduction of white blood cells. The present compounds are also usefulin the development of other compounds that bind to G-CSFR, because thecompounds provide important structure-activity relationship (SAR)information that facilitates that development.

Moreover, based on the ability-to bind to G-CSFR and related receptors,a compound of the invention can be used as a reagent for detecting aG-CSF receptor or related receptor on living cells, fixed cells, inbiological fluids, in tissue homogenates, in purified, naturalbiological materials, etc. For example, by labeling a compound of theinvention, one can identify a cell expressing G-CSFR on its surface. Inaddition, based on it ability to bind a G-CSFR, a compound of theinvention can be used in in situ staining, FACS (fluorescence-activatedcell sorting), Western blotting, ELISA (enzyme-linked immunoadsorptiveassay), etc. In addition, because of its ability to bind to a G-CSFR, acompound of the invention can be used in receptor purification or inpurifying cells expressing G-CSFR on the cell surface (or insidepermeabilized cells).

A compound of the invention can also be utilized as a commercialresearch reagent for various medical research and diagnostic uses. Suchuses include but are not limited to: (1) use as a calibration standardfor quantitating the activities of candidate G-CSFR antagonists oragonists in a variety of functional assays; (2) use as a blockingreagent in random peptide screening, i.e., in searching for new familiesof G-CSFR peptide ligands; (3) use in the co-crystallization withG-CSFR, i.e., a compound of the invention will allow formation ofcrystals bound to G-CSFR, enabling the determination of receptor/peptidestructure x-ray crystallography; (4) use in inhibiting or decreasing theproliferation and growth of G-CSF-dependent cell lines; and (5) otherresearch and diagnostic applications wherein the action of G-CSFR is tobe mimicked, and the like.

A compound of the invention can also be administered to a warm bloodedanimal, including a human, to treat a disease that would benefit fromthe ability of a compound to mimic the effects of G-CSF in vivo. Thus,the present invention encompasses methods for treating a patient whowould benefit from a G-CSFR modulator, comprising administering to thepatient a therapeutically effective amount of a compound of theinvention to activate G-CSFR. For example, a compound of this inventionwill find use in the treatment of diseases such as a depressedneutrophil count. Although attributable to a myriad of causes, adepressed neutrophil count is commonly associated with chemotherapy,AIDS and pneumonia (particularly community-acquired pneumonia). Thus, itis preferred that a compound of the present invention be used to treat adepressed neutrophil count selected from the group consisting ofchemotherapy-induced neutropenia, AIDS-induced neutropenia andcommunity-acquired pneumonia-induced neutropenia.

In addition, the invention encompasses methods for treating a patientwho would benefit from a G-CSFR modulator, comprising administering tothe patient a therapeutically effective amount of a compound of theinvention that antagonizes the action of G-CSF to the G-CSFR in vivo.For example, these receptor antagonists are administered prior to andduring chemotherapy to confer chemoprotection to the neutrophilprogenitor cells by preventing their proliferation in the presence ofcytotoxic drugs. Once chemotherapy administration is suspended, theadministration of the chemoprotective G-CSFR antagonists is alsosuspended thereby allowing the patient's endogenous G-CSF to stimulateproliferation. Alternatively, the neutrophil progenitor cells may be“rescued” by administration of G-CSF or by a G-CSF agonist, e.g., acompound of the present invention having G-CSF agonist activity.

Accordingly, the invention includes pharmaceutical compositionscomprising, as an active ingredient, at least one of the -compounds ofthe invention in association with a pharmaceutical carrier or diluent.The composition can be administered by oral, parenteral (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous) injection,transdermal (either passively or using iontophoresis orelectroporation), or transmucosal (nasal, vaginal, rectal, orsublingual) routes of administration, or using bioerodible inserts, andcan be formulated in dosage forms appropriate for each route ofadministration.

Solid dosage forms for oral administration include capsules, tablets,pills powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, an additional substance other than aninert diluent, e.g., a lubricating agent such as magnesium stearate. Inthe case of capsules, tablets, and pills, the dosage forms may alsocomprise a buffering agent. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions and syrups, with theelixirs containing an inert diluent commonly used in the art, such aswater. These compositions can also include one or more adjuvants, suchas a wetting agent, an emulsifying agent, a suspending agent, asweetening agent, a flavoring agent or a perfuming agent.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain one or more adjuvants such as a preserving agent, a wettingagent, an emulsifying agent and a dispersing agent. The dosage forms maybe sterilized by, for example, filtration through a bacteria-retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured using sterile water, or some other sterileinjectable medium, prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance, anexcipient such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with one or morestandard excipients well known in the art.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient is such that a suitable dosage form is obtained. The selecteddosage depends upon the desired therapeutic effect, the route ofadministration, the duration of the treatment desired, and other factorswell known to those skilled in the art. Generally, dosage levels ofbetween 0.001 to 10 mg/kg of body weight daily are administered tomammals.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples which follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications -within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entirety.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toprepare and use the compounds disclosed and claimed herein. Efforts havebeen made to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.) but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. and pressure is at or near atmospheric.

Standard peptide synthetic methods were used, and solid phase reactionswere carried out at room temperature. Unless otherwise indicated, allstarting materials and reagents were obtained commercially, e.g., fromAldrich, Sigma and ICN, and used without further purification. Standardcell culture and cell harvesting procedures were used.

Also, in these examples and throughout this specification, theabbreviations employed have their generally accepted meanings, asfollows:

-   -   Ac=acetyl    -   BSA=bovine serum albumin    -   DMSO=dimethyl sulfoxide    -   DTT=dithiothreitol    -   HPLC=high pressure liquid chromatography    -   MBP=maltose binding protein    -   PBS=phosphate-buffered saline    -   SDS PAGE=sodium dodecyl sulfate polyacrylamide gel        electrophoresis    -   TCEP=tris(2-carboxyethyl)phsophine    -   TFA=trifluoroacetic acid    -   Tris=tris[hydroxymethyl]aminomethane

EXAMPLES 1-34 G-CSF Competition Binding Assays

The peptides of Table 1 were synthesized using standard techniques andwere subsequently evaluated to identify whether the peptides exhibitedspecific and/or competitive binding.

Specific binding is binding of a ligand to a specific receptor, asopposed to non-specific binding that is mediated by non-specificinteractions. Specific binding may be measured by subtraction of thenon-specific binding (measured in the presence of saturatingconcentrations of unlabeled ligand) from the total binding (measured inthe absence of saturating amounts of ligand). Typically, the unlabeledligand used was a variant of G-CSF in which the cysteine normally foundat position 17 was converted to serine (CS17).

Determination of competitive binding was also carried out for a numberof peptides. Briefly stated, G-CSFR was purified using standardtechniques. The receptor was then immobilized in microtiter plate wellsthat were coated with acid-treated antibody (Ab179) specific for a siteon G-CSFR not involved with G-CSF binding. Separately, ¹²⁵I was coupledto the natural ligand G-CSF using techniques well known in the art. Testpeptides were added to receptor-coated wells and allowed to bind toimmobilized receptor for approximately 30 minutes. ¹²⁵I labeled G-CSFwas then introduced to the wells and incubated overnight at 4° C.Unbound ¹²⁵I labeled G-CSF was removed by washing the plate severaltimes followed by measuring the amount of radioactivity that remained ineach well using conventional techniques. If no reduction in the amountof bound ¹²⁵I labeled G-CSF was detected, the peptide did not competefor binding to the receptor. Alternatively, if reduced amounts or no¹²⁵I labeled G-CSF was detected, the peptide did compete. Non-positiveresults for a particular peptide are not dispositive of that peptide'sactivity: the peptide may exhibit binding under conditions differentfrom those tested.

The results of these assays reveal important information about thestructure activity relationship for peptide and peptide mimetics of theinvention to the G-CSF receptor. TABLE 1 Ex. Specific Competitive No.Sequence Binding ? Binding ? 1 CAGEVMHMCC (SEQ ID NO: 8) Yes Yes 2CNREIEAMCC (SEQ ID NO: 9) Yes Yes 3 CADEVMHFCC (SEQ ID NO: 10) Yes Yes 4CDVWQLFDRC (SEQ ID NO: 25) Yes Yes 5 CSFVQLNSIC (SEQ ID NO: 26) Yes Yes6 CVPWMFYDLC (SEQ ID NO: 29) Yes No 7 CDPWMFYDLC (SEQ ID NO: 30) Yes No8 CQRAGYMLAC (SEQ ID NO: 44) No No 9 CHANPVWGEC (SEQ ID NO: 45) No No 10CTWTDLESVY (SEQ ID NO: 433) No No 11 CFWSDWGQTC (SEQ ID NO: 46) No No 12CPDWYQSYMC (SEQ ID NO: 34) Yes Yes 13 CPHWTSYYMC (SEQ ID NO: 47) Yes Yes14 CACMLRVVHC (SEQ ID NO: 43) Yes Yes 15 CETLCGACFC (SEQ ID NO: 44) NoNo 16 SNESGWVWLP (SEQ ID NO: 110) Yes No 17 EQSNSGWVWV (SEQ ID NO: 111)Yes No 18 SRTESGWVWT (SEQ ID NO: 112) Yes No 19 QRANSGWVWV (SEQ ID NO:113) Yes No 20 DYDNSGWVWH (SEQ ID NO: 114) Yes No 21 ETWGERDWFC (SEQ IDNO: 133) Yes Yes 22 STAERLWFCG (SEQ ID NO: 135) Yes Yes 23 YETAERSYFC(SEQ ID NO: 119) Yes Yes 24 ADNAERGWFC (SEQ ID NO: 137) Yes Yes 25QSNSEREWFC (SEQ ID NO: 138) Yes Yes 26 STSERAWFCG (SEQ ID NO: 139) YesYes 27 ASWSERGWFC (SEQ ID NO: 140) Yes Yes 28 ELSSEREWFC (SEQ ID NO:141) Yes Yes 29 DMQGERGWFC (SEQ ID NO: 142) Yes Yes 30 DMVYAYPPWS (SEQID NO: 155) Yes No 31 DEMVYTVPYW (SEQ ID NO: 156) Yes Yes 32 HTTNEQFFMC(SEQ ID NO: 434) Yes Yes 33 DTWLELESRY (SEQ ID NO: 435) Yes No 34DWQKTIPAYW (SEQ ID NO: 437) Yes Yes

EXAMPLES 35-73 G-CSF Radioligand Binding Assays

The peptides of Table 2 were synthesized using standard techniques andwere subsequently evaluated to determine their binding affinities toG-CSFR.

Streptavidin-coated scintillation proximity assay (SPA) beads (Amersham)were mixed with biotinylated anti-receptor immobilizing antibody (Ab179)followed by incubation with soluble G-CSFR harvest. Receptor-coated SPAbeads were washed twice in PBS/0.1% BSA and distributed to wells of awhite polystyrene 96-well microtiter plate (Packard). Serial dilutionsof peptide or peptide mimetic were mixed with a constant amount of ¹²⁵Ilabeled G-CSF (10⁵ cpm; 1290 Ci/mmol) in PBS/0.1% BSA, added to wellscontaining receptor-coated SPA beads, and incubated overnight at 4° C.The binding of radiolabeled G-CSF to the receptor-coated SPA bead bringsthe isotope in close proximity to the scintillant, which allows theemitted radiation to stimulate the scintillant to emit light. Anyunbound radiolabeled ligand is not in close enough proximity to thescintillant to allow such energy transfer and hence no signal isgenerated. The amount of ¹²⁵I labeled G-CSF that was bound atequilibrium was measured by counting the plate in a TopCount (Wallac)microtiter plate luminometer. The assay is conducted over a range ofpeptide concentrations and the results are graphed such that the y-axisrepresents the amount of bound ¹²⁵I labeled G-CSF and the x-axisrepresents the concentration of peptide or peptide mimetic. One candetermine the concentration at which the peptide or peptide mimetic willreduce by 50% (IC₅₀) the amount of ¹²⁵I labeled G-CSF bound toimmobilized G-CSFR. The dissociation constant (K_(d)) for the peptideshould be similar to the measured IC₅₀ using the assay conditionsdescribed above.

The peptides along with their corresponding IC₅₀ values are shown inTable 2. IC₅₀ values are indicated symbolically by the symbols “−”, “+”,and “++”. For examples, those peptides which showed IC₅₀ values inexcess of 200 uM are indicated with a “−”. Those peptides which gaveIC₅₀ values of less than or equal to 200 uM are given a “+”, while thosewhich gave IC₅₀ values of 500 nM or less are indicated with a “++”.Those peptides, which gave IC₅₀ values at or near the cutoff point for aparticular symbol, are indicated with a hybrid designator, e.g., “±”.The peptides for which IC₅₀ values were not determined are listed as“N.D.”.

The results of these assays reveal important information about thestructure-activity relationship for peptide and peptide mimetics of theinvention to the G-CSF receptor. TABLE 2 Ex. No. Sequence IC₅₀ 35NH₂-EQSNSGWVWV-CONH₂ (SEQ ID NO: 111) + 36 NH₂-STAERLWFCG-CONH₂ (SEQ IDNO: 135) − 37

(SEQ ID NO: 135)  (SEQ ID NO: 135) + 38 NH₂-QSNSEREWFC-CONH₂ (SEQ ID NO:138) − 39

(SEQ ID NO: 138)  (SEQ ID NO: 138) − 40 NH₂-QSNSEREWFCG-CONH₂ (SEQ IDNO: 149) − 41

(SEQ ID NO: 149)  (SEQ ID NO: 149) − 42 Ac-ESGWVW-CONH₂ (SEQ ID NO: 470)− 43 Ac-NSGWVW-CONH₂ (SEQ ID NO: 471) − 44 Ac-SGWVW-CONH₂ (SEQ ID NO:472) − 45 NH₂-EQSNSGWVWVGGGGC-CONH₂ (SEQ ID NO: 101) + 46

(SEQ ID NO: 101)  (SEQ ID NO: 101) + 47 CESRLVECSRM (SEQ ID NO: 462) +/−48 LAHCLLRLEECAAG (SEQ ID NO: 460) +/− 49 ALLMCESKLAECARAR (SEQ ID NO:450) +/− 50

(SEQ ID NO: 339)  (SEQ ID NO: 339) + 51 DLWYLESKLEECARRCNG (SEQ ID NO:340) + 52

(SEQ ID NO: 475)  (SEQ ID NO: 475) + 53 LLDICELKLQECARRCN (SEQ ID NO:208) ++ 54 GGGLLDICELKLQECARRCN (SEQ ID NO: 209) ++ 55GRTGGGLLDICELKLQECARRCN (SEQ ID NO: 210) ++ 56LGIEGRTGGGLLDICELKLQECARRCN (SEQ ID NO: 211) ++ 57 LLDICELKLQECARRAN(SEQ ID NO: 343) + 58 LLDICELKLQEAARRCN (SEQ ID NO: 212) + 59Biotin-LLDICELKLQECARRAN (SEQ ID NO: 343) + 60 Biotin-KLLDICELKLQEAARRCN(SEQ ID NO: 213) + 61 LLDIAELKLQECARRCN (SEQ ID NO: 463) + 62Biotin-KLLDIAELKLQECARRCN (SEQ ID NO: 464) + 63Biotin-KGGGMLAERKAEERRWFNTHGRE (SEQ ID NO: 490) + 64

(SEQ ID NO: 377)  (SEQ ID NO: 378) +/− 65

(SEQ ID NO: 380)  (SEQ ID NO: 381) N.D. 66H₂N-KSTGGLTAERDAEKRRWLLTHGGE-COOH (SEQ ID NO: 491) − 67

(SEQ ID NO: 465)  (SEQ ID NO: 465) + 68

(SEQ ID NO: 466)  (SEQ ID NO: 467) − 69

(SEQ ID NO: 468)  (SEQ ID NO: 469) − 70 YLELCQLRLEECARQFN (SEQ ID NO:282) + 71 CGCHVSPVQIKALC (SEQ ID NO: 198) + 72 GCHVSPVQIKALC (SEQ ID NO:199) − 73 HELCETYADWLGCVEW (SEQ ID NO: 76) N.D.

EXAMPLES 74-81 Cell Proliferation and Luminescence Assays

The bioactivity of selected peptides of the invention was measured incell-based assays. Murine NFS-60 cells proliferate in the presence ofG-CSF in a dose dependent manner and were used in standard cellproliferation assays that are well known in the art. Murine IL-3dependent Ba/F3 cells were co-transfected with expression vectorsencoding the full length human G-CSFR and a luciferase reporter genecontrolled by the fos promoter. The Ba/F3 G-CSFR reporter cell line isnot only dependent on the presence of G-CSF for proliferation, but alsoproduces luciferase in response to the addition of G-CSF in a dosedependent manner. The parental, untransfected cell line does not respondto G-CSF or produce luciferase, but remains IL-3 dependent.

Reporter cell assays were performed on the above cell line usingpeptides of the invention. The cells were maintained in completeRPMI-1640 media containing 10% fetal calf serum, 2 mM L-glutamine, 1×antibiotic-antimycotic solution (Life Technologies), and 10% WEHI-3conditioned media (source of murine IL-3). For reporter assays, cellswere starved overnight in medium which lacks WEHI-3 to reduce luciferaseexpression to background levels. The cells were then washed twice inPBS, resuspended in media which lacks WEHI-3 conditioned media, andadded to wells of a 96-well microtiter plate containing dilutions ofpeptide or G-CSF at 5 x 104 cells/well. Plates were incubated for 2hours at 37° C. in a humidified 5% CO₂ incubator and luciferase activitywas measured by the addition of luciferin (LucLite—Packard Biosciences)to each well. The plates were read in a TopCount (Wallac) microtiterplate luminometer.

To measure the ability of selected peptides of the invention to blockG-CSF mediated receptor activation, dilutions of peptide were combinedwith Ba/F3 G-CSFR reporter cells as described above. After a 30-minuteincubation at 37° C., G-CSF was added to each well. The cells wereincubated for 2 hours at 37° C. and the amount of luciferase producedwas measured as described above.

The following seven peptides were tested for bioactivity: Ex. 74NH₂-EQSNSGWVWV-CONH₂; (SEQ ID NO: 111) Ex. 75 NH₂-STAERLWFCG-CONH₂; (SEQID NO: 135) Ex. 76 NH₂-STAERLWFCG-CONH₂; (SEQ ID NO: 135)NH₂-STAERLWFCG-CONH₂; (SEQ ID NO: 135) Ex. 77 QLETCVLKLEECARRCN; (SEQ IDNO: 315) Ex. 78 LLDICELKLQECARRCN; (SEQ ID NO: 208) Ex. 79PLFSCELKKQECARRCN; (SEQ ID NO: 323) and Ex. 80 DLWYLESKLEECARRCN. (SEQID NO: 338)

Examples 74, 75, and 76 showed antagonist activity at highconcentrations in cell-based assays using NFS-60 cells. The stability ofExample 74 in cell culture medium was tested by overnight incubation inNFS-60-conditioned medium; no loss of activity was observed, indicatingthat the peptide is stable to degradation under these conditions.

Examples 77, 78, 79, and 80 showed cell proliferation activity whenfused to the carboxy-terminus of the maltose binding protein (MBP). TheMBP fusion protein of Example 78 in particular showed high affinity in abinding competition assay with ¹²⁵I-GCSF (IC₅₀ ˜10 nM) and activity in aBa/F3 G-CSFR cell proliferation assay (maximal activity at 100 nM).Parental Ba/F3 cells and Ba/F3 cells expressing the human thrombopoietinreceptor did not proliferate in response to this fusion protein. Westernblot analysis of the fusion protein revealed both monomeric and dimericspecies, however the G-CSFR preferentially binds the dimeric molecule.This is true for most of the MBP fusions tested. Presumably the fusionprotein is dimerized through intermolecular disulfide bonds betweencysteine residues present in the peptide sequence. Cleavage of thepeptide from the carboxy terminus of MBP using Factor Xa caused thepeptide to lose its bioactivity while retaining its binding activity.

The Ba/F3 G-CSFR reporter cell line was used to measure the potency of:Ex. 81 LLDICELKLQECARRCN (SEQ ID NO: 208)and other possible G-CSF receptor antagonists.

Ligand mediated G-CSF receptor activation in these cells results in theexpression of luciferase, providing a detectable biological signal.Ba/F3 G-CSFR reporter cells responded to the addition of G-CSF in a dosedependent manner (FIG. 2). The addition of increasing concentrations ofpeptide from Example 81 inhibit this G-CSF response, indicating that thepeptide is a G-CSFR antagonist (FIG. 3).

EXAMPLE 82 Characterization of the Dimer Form of AF15846

The peptide AF15846, i.e., LLDICELKLQECARRCN (SEQ ID NO: 208), was understudy as a G-CSF antagonist for chemoprotection againstchemotherapy-induced neutropenia. The peptide monomer contains three Cysresidues with a mass of 2020.4 (average). This peptide is not active asa monomer but must be oxidized, putatively to a dimer form, foractivity.

Monomer vs. Dimer Forms of AF15846:

AF15846 that had been oxidized in 50 mM Tris, pH 8.0 for 48 hours wasdiluted with PBS, then injected onto a Superdex peptide gel filtrationcolumn equilibrated in PBS at 0.75 mL/min. The results of thischromatography indicated that most of the peptide was in dimer form,with small amounts of monomer remaining (not shown). In contrast, AF15846 that had been stored in acid and then diluted with PBS directlyprior to injection onto the peptide column eluted predominantly as amonomer. Some dimerization apparently occurred either during storage orduring the short period the peptide was at neutral pH prior to andduring size exclusion chromatography. Oxidized peptide also eluted muchlater from a cation exchange column run in salt gradients at low pH,consistent with dimer formation (not shown).

Reverse Phase HPLC Assay for Oxidation of AF15846:

AF 15846 was oxidized by incubation in 50 mM Tris, pH 8.0, for 16 to 48hours. Reverse phase HPLC methods using a Vydac 25 cm C-18 column and0.1% TFA/acetonitrile buffers were developed to separate the oxidizeddimer from unoxidized monomer, and to separate several differentdimerized peptide structures. While both high pH reverse phase andcation exchange chromatography were also investigated, low pH reversephase separation on a 25 cm column provided the best separation of themany oxidized forms of the peptide (not shown). The dimer species elutefrom the column with earlier retention times than do the monomerspecies. Samples of oxidized AF15846 were re-reduced with DTT to confirmthe elution order. One additional piece of evidence for the formation ofintermolecular dimers comes from the fact that when oxidation wascarried out at low (0.25 mg/mL) concentrations of peptide, the reactionapparently did not go to completion.

Oxidation of AF15846 Under Various Conditions:

AF1 5846 was incubated for 48 hours in 50 mM Tris, pH 8, 20% DMSO inwater, 20 mM potassium phosphate, pH 3, or 0.1% TFA at room temperature.Aliquots of each sample were taken at various time points. Oxidation ofthe monomer peptide in Tris resulted in the presence of one major plusone minor oxidized species after several hours. In contrast, oxidationof the peptide in 20% DMSO in water resulted in a complex mixture ofoxidized species, even after the 48 hour incubation. Some oxidation ofthe peptide was observed even at acidic pH, although to a much lesserextent than that observed with either Tris or DMSO as the oxidant.

Activity of Oxidized AF15846 Fractions:

Several fractions containing oxidized AF15846 resulting from treatmentunder the conditions described above were collected subjected to testingin two assays: an ¹²⁵I-G-CSF competition binding assay and an ELISAformat competitive G-CSF receptor-binding assay. In both cases fractionscorresponding to the predominant Tris-oxidized species exhibited thehighest activity. The activity of selected fractions in the ¹²⁵I-G-CSFcompetition binding assay is shown in FIG. 4. While speciescorresponding to the monomer peptide were inactive, matrix-assistedlaser desorption/ionization mass spectrometry (MALDI-MS) confirmed thatthe active, Tris-oxidized species was a peptide dimer.

Determination of the Disulfide Structure of the Active Oxidized Form ofAF15846:

It was hypothesized that the active form of AF 15846 would contain oneintrachain disulfide per peptide monomer and one interchain peptidedimer the three possibilities for this type of structure are shown belowH₃N⁺-LLDICELKLQECARRCN-COO⁻ (SEQ ID NO:208)          |      |   |         |          |      {overscore (|   |)}H₃N⁺-LLDICELKLQECARRCN-COO⁻; (SEQ ID NO:208) H₃N⁺-LLDICELKLQECARRCN-COO-(SEQ ID NO:208)          |      |   |                     |         {overscore (|      |)}   | H₃N⁺-LLDICELKLQECARRCN-COO⁻; and(SEQ ID NO:208) H₃N⁺-LLDICELKLQECARRCN-COO⁻ (SEQ ID NO:208)         |      |   |                 |          {overscore(|      |   |)} H₃N⁺-LLDICELKLQECARRCN-COO⁻. (SEQ ID NO:208)To determine if one of these structures was present in the active formof AF15846, aliquots of Tris-oxidized AF15846 (not HPLC purified) weredigested with trypsin and subjected to reverse phase HPLC. Trypsindigestion was carried out using an immobilized enzyme column fromPerseptive Biosystems. Digestion was carried out in 25 mM Tris, pH 8, 5mM CaCl₂. Fractions were eluted from the column directly into 0.1% TFAto lower the pH and minimize disulfide scrambling. The resulting trypticfragments were separated by reverse phase HPLC and analyzed by MALDImass spectrometry and Edman sequencing. In addition, an aliquot of thedigest was analyzed by electrospray liquid chromatography/massspectrometry (LC/MS). MALDI MS and sequencing of the tryptic peptidesindicated the presence of peptides corresponding to disulfide bondsbetween Cys-5 and Cys-5, as well as between Cys12 and Cys-12. Thisfinding indicated that there were two interchain disulfide bonds betweenpeptide monomers. This result was confirmed by the LC/MS data (FIG. 5),which identified peptides identical to those found by MALDI MS. Thetyptic peptides are labeled, beginning with the first residue, i.e.,Lys, as follows: T1=residues 1-8; T2=residues 9-14; T1,2=residues 1-14;T2,3=residues 9-15; and “+” indicates a disulfide linkage betweenpeptides. However, an additional minor species was evidently present, asa peptide corresponding to a disulfide bond between Cys-5 and Cys-12,which could be either an intrachain or an interchain disulfide, was alsoseen, albeit at a lower level.

To confirm that the active species contained at least two interchaindisulfides, an aliquot of the HPLC-purified, Tris-oxidized AF15846 shownto be active in competition assays was also digested with trypsin. Theprofile of the purified material was compared to that of theunfractionated Tris oxidation product (FIG. 6, same labeling as in FIG.5). The HPLC profile indicates that the purified material is lacking apeptide corresponding to a Cys-5 to Cys-12 disulfide-linked fragment.This indicated that the active species contains two interchain disulfidebonds. However, the oxidation state of the remaining Cys-16 in eachmonomer was not determined.

The oxidized peptide was also reacted with N-ethylmaleimide (NEM) at 37°C. for 1 hour in 100 mM ammonium acetate, pH 4.1 to see if any free Cysresidues remained in the molecule. If this were the case, treatment withthe alkylating reagent would result in a shift of the HPLC retentiontime. Upon incubation with NEM, no such shift was seen (FIG. 7). Incontrast, when the oxidized peptide was incubated with the disulfidespecific reducing agent TCEP, also in ammonium acetate, a shift to alater retention time, consistent with reduced peptide, was found. Thereduced peptide was modified with NEM to produce a peptide that elutedeven later than the reduced form. These data indicate that all six Cysresidues in the AF15846 active dimer are involved in disulfide bonds.Since previous results showed that Cys-5 is linked to Cys-5 and Cys-12is linked to Cys-12, it seems apparent that the remaining two Cysresidues at position 16 of the monomer are also involved in aninterchain disulfide bond.

To obtain further information about the disulfide bond structure inactive AF15846, the peptide was digested with Lys-C in 50 mM Tris pH7.0/30% acetontrile. The profile of this digest is shown in FIG. 8. Fourmajor peaks are seen. The first peak corresponds to a dimer of residues9-17, as indicated by the MALDI MS spectrum of this fraction. See FIGS.9A and 9B. However, it is not possible to tell with this technique ifall four Cys residues are involved in disulfide formation. The last peakcontains a dimer of residues 1-8. The remaining two peaks representintact peptide (22 min) and an artifact peak. This second digest clearlyindicates that the peptide dimerizes into a parallel structure.

This three parallel interchain disulfide structure, indicated below, isdifferent than that originally predicted. Note that the arrows representsites of cleavage by trypsin.             ↓     ↓ NH₃⁺-LLDICELKLQECARRCN-COO⁻ (SEQ ID NO:208)         |      |   | NH₃⁺-LLDICELKLQECARRCN-COO⁻ (SEQ ID NO:208)             ↑     ↑                AF15846 (dimer form)Incubation of the oxidized peptide at 37° C. at higher pH apparentlyresulted disulfide scrambling and/or degradation of the peptide ascontrol peptide fractions incubated at pH 6.0 or pH 7.5 in parallel withNEM-treated fractions exhibited complex HPLC patterns after incubation.It was necessary to drop to pH 4.1 to obtain clean profiles upon NEMtreatment.A Bioassay for Determining Activity of G-CSF Antagonists:

A biosassay was used to measure the potency of AF15846 and otherpossible G-CSF receptor antagonists. This bioassay utilizes a Ba/F3 cellline containing the rhGCSF receptor and a c-fos promoter/luciferase geneconstruct (Ba/F3/rhGCSF-R/pFos-lcf). Competent binding of a ligand tothe receptor results in expression of lucifierase as the biologicalreadout. Addition of AF15846 to the assay results in the dose-responsecurve shifting to higher concentrations, indicating that the peptide isinhibiting the binding of G-CSF to the expressed receptor (FIGS. 10A and10B). Conversely, the inclusion of various levels of peptide in theassay causes an increase in the amount of G-CSF required to produce asignal, also indicating that the peptide inhibits G-CSF binding (FIG.11).

1-161. (CANCELED)
 162. A compound comprising a peptide chainapproximately 12 to 40 amino acids in length that binds to G-CSFR andcontains a sequence of amino acids of formula (V) (V) CX^(IV) ₁X^(IV)₂X^(IV) ₃X^(IV) ₄X^(IV) ₅X^(IV) ₆X^(IV) ₇X^(IV) ₈X^(IV) ₉X^(IV) ₁₀C (SEQID NO: 5) wherein each amino acid is indicated by standard one-letterabbreviation, and wherein X^(IV) ₁ is E, G, P, N, R, T, W, S, L, H, A, Qor Y; X^(IV) ₂ is S, T, E, A, D, G, W, P, L, N, V, Y, R or M; X^(IV) ₃is R, Y, V, Q, E, T, L, P, S, K, M, A or W; X^(IV) ₄ is L, M, G, F, W,R, S, V, P, A, D, C or T; X^(IV) ₅ is V, T, A, R, S, L, W, C, I, E, P,H, F, D or Q; X^(IV) ₆ is E, Y, G, T, Q, M, S, N, A or P; X^(IV) ₇ is C,V, D, G, L, W, E, V, I, S, M or A; X^(IV) ₈ is S, Y, A, W, P, V, L, Q,G, K, F, I, E or D; X^(IV) ₉ is R, W, M, D, H, V, G, A, Q, L, S, E or Y;X^(IV) ₁₀ is M, L, I, S, V, P, W, F, T, Y, R, or Q; and wherein saidcompound does not comprise sequence LLDICELKLQECARRCN (SEQ ID NO: 208).163. The compound of claim 162, wherein X^(IV) ₁ is E, X^(IV) ₂ is S orA, X^(IV) ₃ is R, X^(IV) ₄ is L, X^(IV) ₅ is V or S, X^(IV) ₆ is E,X^(IV) ₇ is C, X^(IV) ₈ is S, X^(IV) ₉ is R, and X^(IV) ₁₀ is L. 164.The compound of claim 162, wherein the sequence of amino acids isselected from the group consisting of: GGGLLDICELKLQECARRCN (SEQ ID NO:209); GRTGGLLDICELKLQECARRCN (SEQ ID NO: 210);LGIEGRTGGGLLDICELKLQECARRCN (SEQ ID NO: 211); LLDICEELKLQEAARRCN (SEQ IDNO: 212); and KLLDICELKLQEAARRCN (SEQ ID NO: 213).
 165. The compound ofclaim 162, comprising a dimer having the structure of formula (VIII)

wherein R¹ and R² are independently selected from the sequences of aminoacids of formula (V); βA is a β-alanine residue; n1, n2, n3, n4, x and yare independently zero or one with the proviso that the sum of x and yis either one or two; and Lk is a terminal linking moiety selected fromthe group consisting of a disulfide bond, a carbonyl moiety, a C1-12linking moiety optionally terminated with one or two-NH-linkages andoptionally substituted at one or more available carbon atoms with alower alkyl substituent, a lysine residue or a lysine amide.
 166. Thecompound of claim 162, containing a disulfide bond.
 167. The compound ofclaims 162 wherein the N terminus of the peptide is coupled to apolyethylene glycol molecule.
 168. The compound of claim 162 wherein theN terminus of the peptide is acetylated.
 169. The compound of claim 162,wherein the C terminus of the peptide is amidated.
 170. A pharmaceuticalcomposition comprising a therapeutically effective amount of thecompound of any claim 162, in combination with a pharmaceuticallyacceptable carrier.
 171. A method for treating a patient who wouldbenefit from administration of a GCSF modulator, comprisingadministering to the patient a therapeutically effective amount of thecompound of claim
 162. 172. The method of claim 171, wherein the G-CSFmodulator is an agonist for the GCSFR.
 173. The method of claim 171,wherein the patient suffers from a depressed neutrophil count.
 174. Themethod of claim 173, wherein the depressed neutrophil count isassociated with a condition selected from the group consisting ofchemotherapy-induced neutropenia, AIDSinduced neutropenia andcommunity-acquired pneumonia-induced neutropenia.